GENOMICS OF SPERMATOGENIC IMPAIRMENT

描述（由申请人提供）：严重生精失败（SF）影响近1%的男性，约占所有男性不育病例的20%，表现为射精中精子缺乏（少精症）或缺失（无精症）。由于目前超过一半的SF病例没有明确的病因，医生在为SF患者提供有用指导的能力受到严重限制，这些指导涉及使用辅助生殖技术（ART）成功治疗的机会以及他们的ART衍生后代的潜在负面影响。尽管如此，近十年来，我们小组的临床成员已经在男性不育的治疗中常规使用基因检测，因为已知几个Y染色体微缺失是生精失败的诊断，并且是手术取精成功的预后。哺乳动物的精子发生需要超过1000个基因，超过200个小鼠基因敲除显示SF；我们假设超过20%的特发性SF病例是由于单基因或少原性缺陷。我们最近发表了一项基于阵列的初步研究，表明SF的许多遗传原因仍有待发现，DMRT1基因的反复缺失是SF的一个常见的、新的遗传原因。在这个建议中，我们描述的实验将大大扩展这些发现，以确定新的遗传原因SF。我们将对1000名特发性无精子症男性和1000名精子浓度正常的对照男性进行全外显子组测序。有了这些数据，我们将使用特殊的计算和实验试剂来提高我们对重复序列丰富的Y染色体的分析，从而生成遗传变异的综合图谱。我们将使用非靶向变异和基于基因的检测进行全基因组病例对照分析，并通过将变异聚合到途径或基因组注释中来测试许多生物学假设。我们建议使用有效的两阶段设计来复制前1%的相关变异和前5%的相关基因。我们设计了对三种不同形式的SF突变的最大敏感性的I期研究。在阶段I(a)中，我们重新使用作为目标1的一部分生成的对照数据，使用精子数量的数量性状分析来复制中等影响的常见风险因素。在I(b)阶段，我们使用一种极端的表型采样策略，从精子数量分布的顶部和底部的10%中对新患者进行采样，最大限度地提高我们检测极其罕见的大影响单基因/孟德尔变异的能力。在I(c)阶段，我们使用来自原发性卵巢功能不全（POI）女性的现有遗传数据来检测调节女性和男性性腺功能的基因/遗传变异。在I（a,b,c）中至少有一个复制的感兴趣变异将在3000例无精子症病例和3000例对照的中国队列中进行验证。在短期内，我们的发现将提高临床医生的能力

DESCRIPTION (provided by applicant): Severe spermatogenic failure (SF) affects nearly 1% of all men, accounting for approximately 20% of all cases of male infertility, and manifests as either scarcity (oligozoospermia) or absence (azoospermia) of sperm in ejaculate. Physicians are severely limited in their ability to offer useful direction for SF patients pertaining to the chances for successful treatment using assisted reproductive techniques (ART) and potential negative consequences in their ART-derived offspring because more than half of SF cases currently have no identifiable cause. Nonetheless, clinical members of our group have used genetic testing routinely in the management of male infertility for nearly a decade, as several Y chromosome microdeletions are known to be diagnostic of spermatogenic failure, and are prognostic for the success of surgical sperm retrieval. Over 1,000 genes are required for spermatogenesis in mammals, and over 200 mouse knockouts manifest SF; we hypothesize that over 20% of idiopathic SF cases are due to single-gene or oligogenic defects. We have recently published an array-based pilot study demonstrating that numerous genetic causes of SF remain to be discovered, and that recurrent deletions of the gene DMRT1 are a frequent, novel genetic cause of SF. In this proposal we describe experiments that will dramatically expand upon these findings to identify novel genetic causes of SF. We will perform whole exome sequencing of 1000 idiopathic azoospermic men, and 1000 control men with normal sperm concentrations. With these data we will produce an integrated map of genetic variants, using special computational and experimental reagents to enhance our analysis of the repeat-rich Y chromosome. We will perform a genomewide case-control analysis using untargeted variant and gene-based testing, as well as testing numerous biological hypotheses by aggregating variants into pathways or genomic annotations. We propose to use an efficient two-stage design to replicate the top 1% of associated variants and top 5% of associated genes. We have designed the stage I studies for maximum sensitivity to 3 distinct forms of SF mutations. In stage I(a) we re-use the control data generated as part of aim 1 to replicate common risk factors of moderate effect using a quantitative-trait analysis of sperm count. In stage I(b) we use an extreme phenotype sampling strategy to sample new patients from the top and bottom 10% of the sperm count distribution, maximizing our power to detect extremely rare monogenic/Mendelian variants of large effect. In stage I(c) we use existing genetic data from women with primary ovarian insufficiency (POI) to test for genes/genetic variants that modulate gonadal function in women as well as men. Variants of interest that are replicated in at least one of I(a,b,c) will then be taken forward for validation in a Chinese cohort of 3,000 azoospermic cases and 3,000 controls. In the short term, our findings will improve the ability of clinicians to use genetic data to counsel patients and improve patient care. In the long term, characterization of the genetic basis of spermatogenic failure could pave the way for the use of gene therapy strategies in azoospermic men.